Most therapeutic molecules require delivery to a living cell by some means in order to effect a response. Standard therapies include oral administration or other techniques to introduce a treatment molecule into the system. However, even with a therapeutic molecule in the vicinity of a cell, the cell membrane can partially or completely block the uptake ˜f that molecule into the cell itself. To overcome this, many methods have been developed; one such method is the use of electric fields to facilitate passage of the molecules from the extracellular space to the intracellular space.
Scientific research has led to the current understanding that exposure of cells to intense electric fields for brief periods of time temporarily destabilizes membranes. This effect has been described as a dielectric breakdown due to an induced transmembrane potential, and was termed “electroporation,” or “electropermeabilization,” because it was observed that the molecules. that do not normally pass through the membrane gain intracellular access after the cells were treated with electric fields. The porated state was noted to be temporary, with the cells typically remaining in a destabilized state on the order of a few minutes after the cessation of the electrical fields.
The physical nature of electroporation makes it universally applicable. A variety of in vivo procedures utilize this type of treatment to gain temporary access to the cytosol. These include the delivery of drugs to cells within tissues and the delivery of DNA to cells within tissues. A notable example of loading molecules into cells in vivo is electrochemotherapy. The procedure utilizes a drug combined with electric pulses as a means for loading tumor cells with an anticancer drug, and has been performed in a number of animal models and in clinical trials (see, for example, Heller et al., Cancer 77, 964-71, 1996). Also, plasmid DNA has been loaded into rat liver cells (Heller et al., FEBS Left. 389, 225-28, 1996), murine tumors (Niu et al., Cancer Research 59, 5059-63, 1999), rat hepatocellular carcinomas (Heller et al.” Gene Therapy 7,826-29, 2000}, and murine skin in vivo [Heller et al., DNA and Cell Biology20(1}, 21-26, 2001].
The loading of molecules by electroporation in vivo is typically, but not necessarily,
carried out by first exposing the cells (located within a tissue) of interest to the molecule to be loaded. This is accomplished by placing the molecules of interest into the extracellular space by injection, jet injection, transdermal delivery, infusion into tissue or blood vessel, or other means known in the art. The cells are then exposed to electric fields by administering one or more direct current pulses. Pulsed electric fields are normally applied using an electrical generator and electrodes that contact or penetrate a region of tissue, which allows electrical energy to be transmitted to the cells of interest. Electrical treatment is typically, but not necessarily, conducted in a manner that results in a temporary membrane destabilization with minimal cytotoxicity.
The intensity of electrical treatment is described by the magnitude of the applied electric field. This field is defined as the voltage applied to the electrodes divided by the distance between the electrodes. Generally, electric field strengths ranging from 100 to 5000V/cm have been used; this range has been dictated by the need to interfere with the cell membrane to effect the uptake of the molecular species desired. In addition, the field strength is also a function of the type of tissue to be treated, with some requiring higher fields owing to their specific natures.
High field strengths, 100V/cm and greater, were used exclusively in the past. The duration of the applied fields is an important factor, and the relationship between field strength and duration is critical. The current state of the art utilizes high electric field strengths to effect the membrane change and requires pulse durations that are very brief in order to achieve molecular delivery. The concept of very long pulse durations (greater than 100 ms) has heretofore never been used with respect to the field strength, enabling in vivo molecular delivery using almost insignificant electric fields. In fact, the converse was held to be true by practitioners of the art; operating parameters with short-duration high fields being held as the only way to achieve electroporation. The pulsed electric fields used for molecule delivery are generally rectangular in shape; however, exponentially decaying pulses and bipolar pulses have also been used. Molecular loading has been performed with pulse widths ranging from microseconds to milliseconds. The number of pulses delivered typically has ranged from one to eight, with multiple pulses being applied during the course of a treatment.
Work related to the manipulation of the parameters influencing electroporation devices has been the subject of many articles and patents. One such patent, U.S. Pat. No. 6,241,701 to Hoffmann, describes electric field intensities ranging from 25 to 1300 V/cm with times or pulse widths ranging from 10 μs to about 100 ms. The effectiveness of these ranges is described as a correlation between high fields for short duration versus low fields with a preferred longer pulse width. From the statements contained in Hoffmann, one would be led to manipulate the parameters equally with respect to each other, since they are described as being equal in importance. There is no suggestion to vary one parameter namely, the pulse width, to be greater in a nonlinear fashion with respect to the field strength; in addition, there is no teaching to extend the pulse width to any time greater than 100 ms.